Culture-independent PCR-DGGE fingerprinting was used to reveal the bacterial composition and diversity associated with raw milk of mastitis cows from Hokkaido, Japan for the first time. All the mastitis milk samples were diagnosed as solely infection by Coliforms using the classical microbiological method based on on-farm culturing. Our results revealed that the bovine mastitis-associated bacteria were host-specific because community structure varied between each sample. Klebsiella pseudomoniae, Lactococcus lactis Staphylococcus aureus and Escherichia sp were found to be the widely distributed species. Furthermore, more than one mastitis-causing pathogen was found to be present in some mastitis samples. These pathogens may not only act as etiology agents but also play a role in disrupting the natural microbial ecology in mastitis bovine. This finding highlights the limitation of the traditional identification and characterization strategy. Therefore, it is suggested that the methodology applied in this study might be a valuable addition to mastitis control and prevention.
Salmonella typhimurium antigens were displayed on the capsid of a T2 bacteriophage to explore the potential of phage display for an oral vaccine. Segments of the flagellin proteins FliC (H1 antigen) and FljB (H2) were fused to the N-terminal of T2 phage SOC to give two recombinant phages, T2FliCm and T2FljBm. Over 14 days, 19 BALB/c mice were orally administered twice, either with purified recombinant FliCm and FljBm protein, or T2FliCm and T2FljBm with or without host Escherichia coli. Feces were sampled over 10 weeks and examined for phage by plaque assay and for the presence of mucosal IgA by ELISA. Relatively few phages were detected relative to the amount administered (up to 8.21 x 10(3) PFU/g faeces) and none were detected five days after initial administration. The administration of a large number of phages appeared to cause no clinical symptoms. IgA concentration in feces peaked around four weeks after the second administration and subsided after eight weeks. The highest relative titers were observed in the protein group (0.37% for anti-FliCm and 0.22% for anti-FljBm) and the mouse group which received no E. coli (0.33% and 0.35%) despite the theoretical amount of protein contained in a phage dose being at least 80-465 times lower than the protein dose administered. The possibility that the immuno-stimulatory properties of the phage create an adjuvant effect to enhance the immunogenic properties of the displayed proteins is discussed. We conclude that phage may be valuable as a vector for oral vaccines.
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